Project Summary: Organelle size control is a fundamental cell biological problem, and nuclear size is often inappropriately enlarged in cancer cells in a ploidy-independent manner, a change used by pathologists in cancer diagnosis and staging. It is not known if nuclear size changes in cancer are a cause or consequence of disease due to a gap in our knowledge of the mechanisms that regulate nuclear size. My lab addresses fundamental questions about nuclear size regulation using biochemically tractable cytoplasmic extracts that reconstitute nuclear assembly and Xenopus embryos that allow for in vivo functional testing. (1) What mechanisms control nuclear size? Recent progress from my lab has revealed how nuclear import and nuclear lamins contribute to the regulation of nuclear size. To complement candidate approaches to identifying nuclear size effectors, an imaging-based RNAi screen was performed. Results from this screen will be used to dissect novel mechanisms of nuclear size control using Xenopus egg extracts and embryos, focusing on hits enriched in the screen: nuclear structural proteins, regulators of histone and DNA methylation, and vesicular transport proteins. (2) How does cytoplasmic volume influence nuclear size? Using microfluidic-based technologies to encapsulate Xenopus extract in droplets of defined size and shape, my lab recently demonstrated that limiting amounts of a histone chaperone contribute to developmental regulation of nuclear size. (3) What are the physical forces that drive nuclear growth? Having identified multiple regulators of chromatin structure as nuclear size effectors, we hypothesize that intranuclear pushing forces applied to the nuclear envelope allow for protein incorporation into the nuclear lamina, thereby promoting nuclear growth. Using a variety of in vitro approaches, we will test the relative contributions of chromatin structure and nuclear f-actin to nuclear growth and whether intranuclear pushing forces are sufficient to drive nuclear expansion. (4) Elaborating on the microfluidic extract encapsulation approach, we will introduce f-actin, natural cell cycling, and modifications to the droplet cortex. This bottom-up approach to generating synthetic cells with increasingly complex and native attributes will allow us to address questions at the intersection of size control, cytoskeletal organization, and cell cycle timing. (5) How is nuclear size regulated during development and differentiation? To extend our work on Xenopus development to mammalian cells, we have initiated studies with human induced pluripotent stem cells (iPSCs). We find that nuclear morphology and lamin dynamics change significantly during iPSC differentiation, and we will investigate the underlying mechanisms using information gained from the Xenopus system. Our work is bolstered by ongoing productive collaborations that employ diverse interdisciplinary techniques including high-resolution microscopy, RNAi screening, microfluidics, proteomics, and RNA sequencing. Ultimately, the mechanistic information gained from this work will enable experiments to address how nuclear size impacts cell and nuclear function in the context of development, differentiation, and cancer.